HUMAN GLUCAGON-LIKE 1 PEPTIDE RECEPTOR

Abstract: The human GLP-1 receptor is cloned and used in an in vitro assay to screen for compounds that specifically bind to the human GLP-1 receptor, including compounds effective to reduce symptoms of diabetes. The invention includes the assay, the cloned human receptor used in the assay, an isolated human GLP-1 receptor free of other human proteins, and compounds identified through the use of this novel, cloned receptor, which selectively bind the human GLP-1 receptor.

Description: TITLE OF THE INVENTION HUMAN GLUCAGON-LIKE 1 PEPTIDE RECEPTOR BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Sequence of the human GLP-1 receptor. The 463 residue long amino acid sequence deduced from the cDNA sequence is shown in single letter code. The 337 base pair (bp) fragment (hGLP-1 fragment 1) isolated by degenerate PCR is underlined. Three consensus sequences for N-linked glycoslylation are marked with diamonds. Figure 2. Comparison of the human and rat GLP-1 receptor cDNA sequences. Comparison of the human (top) and rat GLP-1 receptors was made using the GCG (Genetics Computer Group) Gap program. The 7 putative transmembrane domains are boxed. Figure 3. Displacement of [ 125j] GLP-1 (7-36) amide binding to transfected COS-7 cells. COS-7 cells (7x1 θ6 cells) were transfected with 20 μg of human GLP-1 receptor cDNA in pcDNAI/neo and membranes prepared and frozen from the cells. 27 μg of membrane protein was incubated with 50 pM [125r GLP-1 (7-36) amide and the indicated concentrations of ligand. Data shown are means +/- S.E.M. of duplicate determinations and are representative of two experiments. Symbols: squares, GLP-1 (7-36) amide; triangles, glucagon; diamonds, gastric inhibitory peptide; circles, secretin. Figure 4. cAMP accumulation in transfected COS-7 cells. COS-7 cells (7x1 θ6 cells) were transfected with 100 μg of human GLP-1 receptor cDNA. Cells were harvested and cAMP accumulation was determined as outlined in the Examples. Data shown are the mean +/- S.E.M. from triplicate determinations from a single experiment and are representative of three experiments. Symbols, triangles, GLP-1 7-36 amide; squares, glucagon. BACKGROUND OF THE INVENTION This is a continuation of U.S. Serial No. 08/104, 517 filed August 9, 1993, now pending. Glucagon-like 1 peptide (GLP-1) is one of several hormones shown to potentiate glucose-induced insulin secretion. Such hormones, known as incretins, are produced in the gut, released in response to a meal, and their interaction with specific receptors on pancreatic islets causes insulin to be secreted in a glucose-dependent manner (H.-C. Fehmann, J. F. Habener, Trends in Endocrinol. and Met. 3, 158-163 (1992)). GLP-1 is produced by postranslational processing of the proglucagon gene in intestinal L cells, through a biologically inactive 37 amino acid form [GLP-1 (1-37)] to either of two biologically active forms, GLP-1 (7-37) and GLP-1 (7-36) amide. These biologically active forms of GLP-1 are the most potent incretins known, with effects on glucose-mediated insulin secretion being seen at concentrations as low as 10 pM. Infusion of GLP-1 (7-36) amide into patients with type II diabetes leads to increased secretion of insulin which occurs in an glucose-dependent fashion (D. M. Nathan, E. Schreiber, H. Fogel, S. Mojsov, J. F. Habener, Diabetes Care 15, 270- 276 (1992); M. Gutniak, C. Orskov, J. J. Hoist, B. Ahren, E. S, New Engl. J. Med. 326, 1316-1322 (1992)). These data suggest that compounds that act via the GLP-1 receptor may be therapeutic in the treatment of type II diabetes. GLP-1 receptors have also been described in lung (G. Richter, R. Goke, B. Goke, A. R., FEBS Lett. 267, 78-80 (1990)), adipose (C. Ruiz-Grande, C. Alarcon, E. Merida, I. Vaverde, Peptides 13, 13-16 (1992)); brain (N. S. Hoosein, G. R.S, FEBS. Lett. 178, 83- 86 (1984)); and a gastric tumor cell line, HGT-1 (A. B. Hansen, C. P. Gespach, G. E. Rosselin, J. J. Hoist, FEBS Lett. 236, 119-122 (1988)). A cDNA encoding a GLP-1 receptor has recently been cloned from rat pancreatic islets (B. Thorens, Proceedings of the National Academy of Sciences U.S.A 89, 8641-8645 (1992)). This receptor has seven putative transmembrane domains and belongs to the superfamily of G protein coupled receptors. The GLP-1 receptor is most homologous to other members of a recently defined subclass of G protein coupled receptors that includes the receptors for glucagon and secretin. The GLP-1 receptor acts via stimulation of adenylyl cyclase to raise intracellular levels of cAMP (Fehmann et al., supra). The present invention pertains to the cloning, expression, and pharmacological characterization of a human GLP-1 receptor from the gastric tumor cell line HGT-1. SUMMARY OF THE INVENTION The human glucagon-like 1 peptide (GLP-1) is cloned, expressed and used in an in vitro assay to screen for compounds that bind to the receptor, including compounds which specifically stimulate or inhibit the activity of the receptor. The invention includes the assay, the cloned receptor used in the assay, an isolated human GLP-1 receptor, cells expressing the cloned receptor, and compounds identified through the use of the cloned GLP-1 receptor which selectively bind to the human GLP-1 receptor, including specific agonists or antagonists of the receptor. The human glucagon-like 1 peptide receptor of the present application was cloned from the gastric tumor cell line HGT-1. The cDNA clone encodes a protein of 463 amino acids. The predicted secondary structure places this receptor within the superfamily of seven transmembrane domain G protein coupled receptors. Transfection of the human GLP-1 receptor into COS -7 cells confers upon them high affinity binding for [ 125 j] GLP-1 (7-36) amide. In membranes prepared from COS-7 cells transfected with the human GLP-1 receptor, the binding of [*25τ] GLP-1 (7-36) amide is inhibited with the rank order of potency GLP-1 (7-36) amide > glucagon > secretin; this is characteristic of a GLP-1 receptor. The human GLP-1 receptor expressed in COS-7 cells is functionally coupled to increases in intracellular cAMP. Incubation of COS-7 cells expressing the human GLP-1 receptor with GLP-1 (7-36) amide gives rise to a 4-fold increase in cyclic AMP over basal levels, with an EC50 of 25 pM. Glucagon is 200-fold less potent than GLP-1 as an agonist at the expressed human receptor. DETAILED DESCRIPTION OF THE INVENTION The human glucagon-like 1 peptide receptor (GLP-1) was identified, cloned and expressed in cell cultures by the instant inventors. A partial coding region for this receptor was generated by polymerase chain reaction technology (PCR). Degenerate oligonucleotides encoding amino acids present in the rat GLP-1 receptor were used to prime PCR reactions using human HGT-1 cDNA as a template. The predicted sized products were cloned and sequenced. Translation of the amplified cDNA yielded an open reading frame encoding a protein approximately 91 % homologous to the rat GLP-1 receptor. This partial sequence was used to obtain a larger cDNA clone from a human HGT-1 library. The remaining receptor cDNA was obtained by a modification of the PCR- RACE (Rapid Amplification of cDNA ends) protocol (M. A. Frohman, M. K. Dush, G. R. Martin, Proc. Natl. Acad. Sci. U.S. A. 85, 8998-9002 (1988)). A cDNA library was made from HGT-1 cDNA and the plasmid pcDNA I as outlined in Examples. PCR was performed using the HGT-1 library and primers to both the partial fragment of the human GLP-1 receptor and pcDNA I. A series of overlapping cDNA fragments were obtained and sequenced. The HGT-1 cDNA library and primers 5' TGGTGGATTCCTGAACTCC 3' (SEQ ID NO: 3) and 5' CCTGTGGTTTCACAAGAAGC 3' (SEQ ID NO: 4) were used in a PCR reaction to generate the complete receptor sequence (Figure 1). The open reading frame included in this sequence encodes a 463 amino acid protein that is approximately 91% identical to the rat GLP-1 receptor sequence (Figure 2). The cloned human GLP-1 receptor, when expressed in mammalian cell lines including but not limited to, COS -7, CHO or L cells, is used to discover ligands that bind to the receptor and alter or stimulate its function. In addition, the cloned GLP-1 receptor enables quantitation of mRNA levels in human tissues, including the pancreas and gastrointestinal system, by RNase protection assays. For these purposes, a complete coding sequence of the receptor is provided. The specificity of binding of compounds showing affinity for the GLP-1 receptor is shown by measuring the affinity of the compounds to membranes obtained from cells tranfected with the cloned GLP-1 receptor and membranes from tissues known to express GLP-1 receptors. Expression of the cloned GLP-1 receptor, screening for compounds that inhibit the binding of radiolabeled GLP-1 (7-36) amide or compounds that stimulate cAMP production in these cells provides a rational way for selection of compounds and discovery of new compounds with predictable pharmacological activities. Once the human receptor is cloned and expressed in a non- human cell line, such as COS-7 cells or CHO cells, the recombinant GLP-1 receptor is free of other human proteins. The membranes from the recombinant cells expressing human GLP-1 receptor are then isolated according to methods well known in the art and may be used in a variety of membrane associated receptor binding assays. One example of such an assay is described by Strader et al., {Proc. Natl. Acad. Sci. USA 84, 4384-4388, 1987). Generally, a compound of interest is used to compete with the binding of a known, quantifiable GLP-1 receptor ligand. Thus, radiolabeled [ 125 I] GLP (7-36) amide or [ H]-GLP may be used for this purpose. Because of the ease of 125j detection, [125j] GLP (7-36) amide is preferred for this purpose. By increasing the amount of unlabeled test compound, the labeled compound is competed off the receptor. From these experiments, IC50 values for each test compound and receptor subtype is determined. In addition, agonist ligands that activate the receptor may be detected by measuring the ability of added compounds to increase cAMP production mediated by the receptor expressed in COS -7 or CHO cells. cAMP can be measured directly by radioimmunoassay or by stimulation of adenylylcyclase in membranes prepared from the cells (Salomon, Y., Landos, C. and Rodbell, M. 1974. Anal. Biochemistry, Vol. 58, 541-548) by methods that are well-known in the art. Thus, according to this invention, a method is provided for identifying compounds specific for the human GLP-1 receptor comprising the following steps: a. Cloning the human glucagon-like 1 peptide (GLP-1) receptor; b. Splicing the cloned GLP-1 receptor into an expression vector to produce a construct such that the GLP-1 receptor is operably linked to transcription and translation signals sufficient to induce expression of the receptor upon introduction of the construct into a prokaryotic or eukaryotic cell; c. Introducing the construct into a prokaryotic or eukaryotic cell which does not express a human GLP-1 receptor in the absence of the introduced construct; d. Incubating cells or membranes isolated from cells produced in Step c with a quantifiable compound known to bind to human GLP-1 receptors, and subsequently adding test compounds at a range of concentrations so as to compete the quantifiable compound from the receptor, such that an IC50 for the test compound is obtained as the concentration of test compound at which 50% of the quantifiable compound becomes displaced from the receptor; and e. Incubating cells or membranes from cells produced in Step c with test compounds in range of concentrations such that an ED50 for the test compound is obtained. The ED50 is defined as the concentration of compound which increases intracellular cyclic AMP to 50% of the maximal quantity of cAMP produced by interaction of the test compound with the human GLP-1 receptor. Whereas GLP-1 has been shown to increase secretion of insulin in diabetic patients, an agonist of the human GLP-1 receptor discovered as described above would be useful in the treatment of diabetes. The following examples are provided to further define the invention without, however, limiting the invention to the particulars of these examples. EXAMPLE 1 Culture of HGT-1 and COS-7 cells HGT-1 cells (cell line C1.19A, a human gastric carcinoma cell line) were obtained from Dr. C. L. Laboisse and cultured as described (Laboisse et al., Cancer Research 42, 1541-1528 (1982)). COS -7 cells were cultured in a manner identical to HGT-1 cells. EXAMPLE 2 Cloning of a partial cDNA human GLP-1 receptor clone Poly A + RNA was isolated from HGT-1 cells using the Fast-Track system (Invitrogen). cDNA was prepared from 5 μg of HGT-1 poly A + RNA by simultaneous priming with random hexanucleotides and oligo dT primers using the Riboclone cDNA synthesis system (Promega). HGT-1 cDNA and degenerate primers based on the rat cDNA sequence (B. Thorens, Proceedings of the National Academy of Sciences U.S. A 89, 8641-8645 (1992)) were mixed in a PCR reaction to amplify a partial fragment of the human GLP-1 receptor cDNA (Figure 1, underlined). The protocol is described below: Degenerate PCR 5 μl lOx PCR buffer from Boehringer Mannheim Biochemicals (BMB). 4 μl 2.5 μM each stock dATP, dCTP, dGTP and dTTP 2 μl HGT-1 cDNA 1 μl 20 μM primer [5' ATG CA(AG) TA(CT) TG(CT) GTN GC 3'; SEQ ID NO:5] 1 μl 20 uM primer [5' AT(AG) TCN GT(AC) TT(AG) CAC AT 3'; SEQ ID NO:6] 0.25 μl (2 units) Amplitaq DNA polymerase (Cetus) 36.75 μl water Reaction conditions: 40 cycles at 95°C, 1 min.; 45°C, 0.5 min.; 72°C, 1 min. The predominant PCR product, a 337 base pair (bp) DNA fragment (hGLP- fragment 1) was cloned into plasmid pCR II using the TA cloning kit (Invitrogen) and transformed into Escherichia coli. INVαF'. Plasmid DNA was isolated and DNA sequence determined by the dideoxy chain termination method. EXAMPLE 3 PCR amplification, cloning, and sequencing of a cDNA encoding the complete human GLP-1 receptor Poly A + RNA was isolated from HGT-1 cells using the Fast-Track system (Invitrogen). cDNA was prepared from 5 μg of HGT-1 poly A + RNA by simultaneous priming with random hexanucleotides and oligo dT primers using the Riboclone cDNA synthesis system (Promega). The cDNA was ligated with non-palindromic BST XI linkers (Invitrogen). Excess linkers were removed by gel-filtration over a cDNA sizing column (Gibco-BRL). For the PCR-RACE protocols the cDNA was ligated into plasmid pcDNA I (Invitrogen) after restriction with BST XL A series of primers were made to sequences in hGLP-fragment 1 (Example 2) and to sequences in pcDNA I. PCR was performed as follows: 5 μl lOx PCR buffer (BMB) 4 μl 2.5 μM each stock dATP, dCTP, dGTP and dTTP 2 μl HGT-1 cDNA 1 μl 20 μM primer from hGLP-fragment 1 1 μl 20 μM primer from pcDNA 1 0.25 μl (2 units) Amplitaq DNA polymerase 36.75 μl water Reaction conditions: 35 cycles at 95°C, 1 min.; 55°C, 0.5 min.; 72°C, 1 min. Aliquots of the PCR reactions were cloned into plasmid pCR II using the TA cloning kit (Invitrogen) and transformed into • E. coli. INV F. Human GLP-1 receptor specific fragments were identified by filter hybridization using [32p] labeled hGLP-fragment 1 as a probe. The following hybridization conditions were employed: 5X SSC (IX SSC is 0.15 M sodium chloride, 0.015 M sodium citrate) 5X Denharts solution (1% Ficoll, 1% polyvinylpyrrolidone) 100 μg/ml salmon sperm DNA 50% formamide Hybridize overnight at 42°C Filters were washed 2 times in IX SSC, 0.1 % SDS at room temperature for 10 min. each, then 2 times in 0.1 X SSC, 0.1% SDS for 20 min. Positive clones were identified by autoradiography. DNA sequence of positive clones was obtained by the dideoxy chain termination method. Using this procedure clones containing DNA sequence from either the 5' or 3' untranslated regions were obtained. A single molecule encoding the entire hGLP-1 receptor sequence was obtained by amplification using the polymerase chain reaction. The following conditions were employed: 5 μl lOx PCR Pfu polymerase buffer (Stratagene). 4 μl 2.5 μM each stock dATP, dCTP, dGTP and dTTP 2 μl HGT-l cDNA 1 μl 20 uM primer 5' TGGTGGATTCCTGAACTCC 3'; SEQ ID NO:7 1 μl 20 uM primer 5' CCTGTGGTTTCACAAGAAGC 3'; SEQ ID NO:8 1 μl (5 units) Pfu polymerase (Stratagene) 5 μl dimethylsulfoxide 29 μl water Reaction mixtures were heated at 96°C for 5 min. and then 35 thermal cycles performed: 1 min. at 95°C, 0.5 min. at 55°C, 2 min. at 72°C. Aliquots of the PCR reactions were blunt end cloned into plasmid pCR-Script SK+ (Stratagene) and transformed into E. coli XL-1 Blue. Human GLP-1 receptor specific fragments were identified by filter hybridization using [32p] labeled hGLP-fragment 1 as a probe. The hybridization conditions employed were those described in Example 3. Filters were washed 2 times in IX SSC, 0.1% SDS at room temperature for 10 min. each, then 2 times in 0.1 X SSC, 0.1% SDS for 20 min. Positive clones were identified by autoradiography. DNA sequence of positive clones was obtained by the dideoxy chain termination method. The DNA sequence obtained is shown in Figure 1. EXAMPLE 4 Expression of the cloned human GLP-1 receptor COS -7 cells were transfected by electroporation with the human GLP-1 receptor cDNA subcloned into the eukaryotic expression vector pcDNA I/neo (Invitrogen). Cells were harvested after 60-72 h. Membranes containing the expressed receptor protein were prepared as described (C. D. Strader et al, Proc. Natl. Acad. Sci. U.S.A. 84, 4384- 4388 (1987). Membranes prepared from the COS-7 cells transfected with the vector containing the human GLP-1 receptor cDNA specifically bound the GLP-1 receptor agonist [ 12 5i] GLP-1 (7-36) amide (Figure 3). Membranes prepared from cells transfected with the vector alone did not specifically bind [* 5τ] GLP-1 (7-36) amide, proving the expression of the human GLP-1 receptor. As shown in Figure 3, GLP-1 (7-36) amide inhibits the binding of [ 12 5i] GLP-1 (7- 36) amide to the receptor with an IC50 of 4 nM. Glucagon, gastric inhibitory peptide, and secretin inhibit [125i] GLP-1 (7-36) amide binding with a potency at least a 100-fold lower, consistent with the identification of the receptor as a GLP-1 receptor. Binding reactions were performed in a final volume of 200 μl of PBS (10 mM sodium phosphate, 1 mM potassium phosphate, 2.7 mM potassium chloride, 137 mM sodium chloride, pH 7.0 ) under the following conditions: 10-25 μg COS-7 membranes prepared from transfected cells 0.1% bovine serum albumin 50 pM [125τ] GLP-1 (7-36) amide 0-1 μM GLP-1 (7-36) amide (or the other compounds listed in the legend to Figure 3) Membranes were incubated at room temperature with shaking for one hour. Membranes were harvested on GF/C filters (Whatman) that had been presoaked in 0.5% polyethylenimine/0.1% BSA. The filters were washed three times with ice-cold PBS and bound radioactivity determined by gamma counting. Data were analyzed using the Inplot program (Graphpad Software). The human GLP-1 receptor is functionally coupled to adenylyl cyclase in transiently transfected COS-7 cells (Figure 4). Incubation of COS-7 cells expressing the human GLP-1 receptor with GLP-1 (7-36) amide leads to a 4-fold increase in cyclic AMP (cAMP) over basal levels. Under identical assay conditions mock transfected COS cells show no significant increase in cAMP over basal levels. GLP-1 (7-36) amide stimulates cAMP accumulation with an EC50 of 25 pM. Glucagon also stimulates cAMP accumulation in COS -7 cells transfected with the human GLP-1 receptor but with a 200-fold decrease in potency compared with GLP-1 (7-36) amide (Figure 4). The decreased potency for glucagon is consistent with its acting via the human GLP-1 receptor. Stimulation of cAMP accumulation in intact COS-1 cells that had been transfected with the hGLP-1 receptor assays were carried out in 120 μl volume of ACC (75 mM Tris pH 7.4, 250 mM sucrose, 12.5 mM magnesium chloride, 1.5 mM ethylenediamine- tetraacetic acid (EDTA), 0.1 mM of the phosphodiesterase inhibitor Ro- 201724) containing the following additions: 50,000 COS-7 cells transfected with the human GLP-1 receptor expression construct; and 0-100 μM GLP-1 (7-36) amide or glucagon Reaction mixtures were incubated for 45 min. at room temperature with shaking. Reactions were terminated by boiling for 3-5 min. cAMP was determined by radioimmunoassay. EXAMPLE 5 Screening Assay: Glucagon-like peptide- 1 receptor mediated increase in intracellular cAMP Transfected cells expressing recombinant human GLP-1 receptor may be used to identify compounds that are agonists for it. This is done by incubating cells with test compounds in range of concentrations such that an ED50 for the test compound is obtained. The ED50 is defined as the concentration of compound which increases intracellular cyclic AMP (cAMP) to 50% of the maximal quantity of cAMP produced by interaction of the test compound with the human GLP-1 receptor. These reactions are carried out in 120 μl volume of ACC containing the following additions: 50,000 COS-7 cells transfected with the human GLP-1 receptor; and Various concentration of test compounds. Reaction mixtures were incubated for 45 min. at room temperature with shaking. Reactions were terminated by boiling for 3-5 min. cAMP was determined by radioimmunoassay. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANTS: Graziano, Michael P. Borkowski, Doreen A. Chicchi, Gary G. Hey, Patricia J. Strader, Catherine D. (ii) TITLE OF INVENTION: Human Glucagon Like 1 Peptide Receptor (iii) NUMBER OF SEQUENCES: 8 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Carty, Christine E. (B) STREET: P.O. Box 2000, 126 E. Lincoln Ave. (C) CITY: Rahway (D) STATE: New Jersey (E) COUNTRY: USA (F) ZIP: 07065 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Carty, Christine E. (B) REGISTRATION NUMBER: 36,099 (C) REFERENCE/DOCKET NUMBER: 19065 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (908) 594-6734 (B) TELEFAX: (908) 594-4720 (C) TELEX: 138825 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1567 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: TGGTGGAATT CCTGAACTCC CCGCCATGGC CGGCGCCCCC GGCCCGCTGC GCCTTGCGCT 60 GCTGCTGCTC GGGATGGTGG GCAGGGCCGG CCCCCGCCCC CAGGGTGCCA CTGTGTCCCT 120 CTGGGAGACG GTGCAGAAAT GGCGAGAATA CCGACGCCAG TGCCAGCGCT CCCTGACTGA 180 GGATCCACCT CCTGCCACAG ACTTGTTCTG CAACCGGACC TTCGATGAAT ACGCCTGCTG 240 GCCAGATGGG GAGCCAGGCT CGTTCGTGAA TGTCAGCTGC CCCTGGTACC TGCCCTGGGC 300 CAGCAGTGTG CCGCAGGGCC ACGTGTACCG GTTCTGCACA GCTGAAGGCC TCTGGCTGCA 360 GAAGGACAAC TCCAGCCTGC CCTGGAGGGA CTTGTCGGAG TGCGAGGAGT CCAAGCGAGG 420 GGAGAGAAGC TCCCCGGAGG AGCAGCTCCT GTTCCTCTAC ATCATCTACA CGGTGGGCTA 480 CGCACTCTCC TTCTCTGCTC TGGTTATCGC CTCTGCGATC CTCCTCGGCT TCAGACACCT 540 GCACTGCACC AGGAACTACA TCCACCTGAA CCTGTTTGCA TCCTTCATCC TGCGAGCATT 600 GTCCGTCTTC ATCAAGGACG CAGCCCTGAA GTGGATGTAT AGCACAGCCG CCCAGCAGCA 660 CCAGTGGGAT GGGCTCCTCT CCTACCAGGA CTCTCTGAGC TGCCGCCTGG TGTTTCTGCT 720 CATGCAGTAC TGTGTGGCGG CCAATTACTA CTGGCTCTTG GTGGAGGGCG TGTACCTGTA 780 CACACTGCTG GCCTTCTCGG TCTTATCTGA GCAATGGATC TTCAGGCTCT ACGTGAGCAT 840 AGGCTGGGGT GTTCCCCTGC TGTTTGTTGT CCCCTGGGGC ATTGTCAAGT ACCTCTATGA 900 GGACGAGGGC TGCTGGACCA GGAACTCCAA CATGAACTAC TGGCTCATTA TCCGGCTGCC 960 CATTCTCTTT GCCATTGGGG TGAACTTCCT CATCTTTGTT CGGGTCATCT GCATCGTGGT 1020 ATCCAAACTG AAGGCCAATC TCATGTGCAA GACAGACATC AAATGCAGAC TTGCCAAGTC 1080 CACGCTGACA CTCATCCCCC TGCTGGGGAC TCATGAGGTC ATCTTTGCCT TTGTGATGGA 1140 CGAGCACGCC CGGGGGACCC TGCGCTTCAT CAAGCTGTTT ACAGAGCTCT CCTTCACCTC 1200 CTTCCAGGGG CTGATGGTGG CCATATTATA CTGCTTTGTC AACAATGAGG TCCAGCTGGA 1260 ATTTCGGAAG AGCTGGGAGC GCTGGCGGCT TGAGCACTTG CACATCCAGA GGGACAGCAG 1320 CATGAAGCCC CTCAAGTGTC CCACCAGCAG CCTGAGCAGT GGAGCCACGG CGGGCAGCAG 1380 CATGTACACA GCCACTTGCC AGGCCTCCTG CAGCTGAGAC TCCAGCGCCT GCCCTCCCTG 1440 GGGTCCTTGC TGCAGGCCGG GTGGCCAATC CAGGAGAAGC AGCCTCCTAA TTTGATCACA 1500 GTGGCGAGAG GAGAGGAAAA ACGATCGCTG TGAAAATGAG GAGGATTGCT TCTTGTGAAA 1560 CCACAGG 1567 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 463 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Ala Gly Ala Pro Gly Pro Leu Arg Leu Ala Leu Leu Leu Leu Gly 1 5 10 15 Met Val Gly Arg Ala Gly Pro Arg Pro Gin Gly Ala Thr Val Ser Leu 20 25 30 Trp Glu Thr Val Gin Lys Trp Arg Glu Tyr Arg Arg Gin Cys Gin Arg 35 40 45 Ser Leu Thr Glu Asp Pro Pro Pro Ala Thr Asp Leu Phe Cys Asn Arg 50 55 60 Thr Phe Asp Glu Tyr Ala Cys Trp Pro Asp Gly Glu Pro Gly Ser Phe 65 70 75 80 Val Asn Val Ser Cys Pro Trp Tyr Leu Pro Trp Ala Ser Ser Val Pro 85 90 95 Gin Gly His Val Tyr Arg Phe Cys Thr Ala Glu Gly Leu Trp Leu Gin 100 105 110 Lys Asp Asn Ser Ser Leu Pro Trp Arg Asp Leu Ser Glu Cys Glu Glu 115 120 125 Ser Lys Arg Gly Glu Arg Ser Ser Pro Glu Glu Gin Leu Leu Phe Leu 130 135 140 Tyr lie lie Tyr Thr Val Gly Tyr Ala Leu Ser Phe Ser Ala Leu Val 145 150 155 160 lie Ala Ser Ala lie Leu Leu Gly Phe Arg His Leu His Cys Thr Arg 165 170 175 Asn Tyr lie His Leu Asn Leu Phe Ala Ser Phe lie Leu Arg Ala Leu 180 185 190 Ser Val Phe lie Lys Asp Ala Ala Leu Lys Trp Met Tyr Ser Thr Ala 195 200 205 Ala Gin Gin His Gin Trp Asp Gly Leu Leu Ser Tyr Gin Asp Ser Leu 210 215 220 Ser Cys Arg Leu Val Phe Leu Leu Met Gin Tyr Cys Val Ala Ala Asn 225 230 235 240 Tyr Tyr Trp Leu Leu Val Glu Gly Val Tyr Leu Tyr Thr Leu Leu Ala 245 250 255 Phe Ser Val Leu Ser Glu Gin Trp lie Phe Arg Leu Tyr Val Ser lie 260 265 270 Gly Trp Gly Val Pro Leu Leu Phe Val Val Pro Trp Gly lie Val Lys 275 280 285 Tyr Leu Tyr Glu Asp Glu Gly Cys Trp Thr Arg Asn Ser Asn Met Asn 290 295 300 Tyr Trp Leu lie lie Arg Leu Pro lie Leu Phe Ala lie Gly Val Asn 305 310 315 320 Phe Leu lie Phe Val Arg Val lie Cys lie Val Val Ser Lys Leu Lys 325 330 335 Ala Asn Leu Met Cys Lys Thr Asp lie Lys Cys Arg Leu Ala Lys Ser 340 345 350 Thr Leu Thr Leu lie Pro Leu Leu Gly Thr His Glu Val lie Phe Ala 355 360 365 Phe Val Met Asp Glu His Ala Arg Gly Thr Leu Arg Phe lie Lys Leu 370 375 380 Phe Thr Glu Leu Ser Phe Thr Ser Phe Gin Gly Leu Met Val Ala lie 385 390 395 400 Leu Tyr Cys Phe Val Asn Asn Glu Val Gin Leu Glu Phe Arg Lys Ser 405 410 415 Trp Glu Arg Trp Arg Leu Glu His Leu His lie Gin Arg Asp Ser Ser 420 425 430 Met Lys Pro Leu Lys Cys Pro Thr Ser Ser Leu Ser Ser Gly Ala Thr 435 440 445 Ala Gly Ser Ser Met Tyr Thr Ala Thr Cys Gin Ala Ser Cys Ser 450 455 460 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TGGTGGATTC CTGAACTCC 19 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CCTGTGGTTT CACAAGAAGC 20 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: ATGCAAGTAC TTGCTGTGCN 20 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: ATAGTCNGTAC TTAGCACAT 20 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7 : TGGTGGATTC CTGAACTCC 19 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: CCTGTGGTTT CACAAGAAGC 20

Claims: WHAT IS CLAIMED IS: 1. An isolated and purified DNA molecule consisting essentially of DNA encoding a human glucagon-like 1 peptide receptor. 2. The DNA molecule of Claim 1 , wherein the DNA coding for the receptor is operably linked to regulatory sequences such that the receptor may be expressed upon introduction into a prokaryotic or eukaryotic cell. 3. The human glucagon-like 1 peptide receptor encoded by the DNA molecule of Claim 1. 4. A cell containing the DNA molecule of Claim 1 , the cell expressing a cloned human glucagon-like 1 peptide receptor. 5. A method for identifying compounds which specifically bind to a human glucagon-like 1 peptide receptor comprising the steps of: (a) cloning the human glucagon-like 1 peptide receptor; (b) splicing the the cloned glucagon-like 1 peptide receptor into an expression vector to produce a construct such that the glucagon-like 1 peptide receptor is operably linked to transcription and translation signals sufficient to induce expression of the receptor upon introduction of the construct into a prokaryotic or eukaryotic cell; (c) introducing the construct into a prokaryotic or eukaryotic cell which does not express a human receptor in the absence of the introduced construct; (d) incubating cells or membranes isolated from cells produced in Step (c) with a quantifiable compound known to bind to human glucagon- like 1 peptide receptor, and subsequently adding test compounds at a range of concentrations so as to compete the quantifiable compound from the receptor; and (e) calculating the relative binding of the test compound to the cells or membranes of Step ( ). 6. The compounds identified by the method of Claim 5. 7. A method of alleviating the effects of diabetes which comprises administering a pharmaceutically effective amount of a compound which specifically binds to the human GLP-1 receptor. 8. The DNA molecule of Claim 1 having the nucleic acid sequence shown in Figure 1. 9. A purified human glucagon-like 1 peptide receptor encoded by the DNA molecule of Claim 8, the purified receptor having the amino acid sequence shown in Figure 1.

Inventors: BORKOWSKI DOREEN A, CHICCHI GARY G, GRAZIANO MICHAEL P, HEY PATRICIA J, STRADER CATHERINE D, BORKOWSKI, DOREEN, A., CHICCHI, GARY, G., GRAZIANO, MICHAEL, P., HEY, PATRICIA, J., STRADER, CATHERINE, D.
Applicants: BORKOWSKI DOREEN A, CHICCHI GARY G, GRAZIANO MICHAEL P, HEY PATRICIA J, MERCK & CO INC, STRADER CATHERINE D, BORKOWSKI, DOREEN, A., CHICCHI, GARY, G., GRAZIANO, MICHAEL, P., HEY, PATRICIA, J., MERCK & CO., INC., STRADER, CATHERINE, D.
Application year: 1994
Main classification: C12N15
IPC codes: A61K38, A61P3, C07H21, C07K14, C12N5, C12N15, C12P21, C12Q1, C12R1